Tissue adhesives continue to evolve as an important technology for the physician, particularly surgeon. Years ago there was little routine use of these substances; however, in the past years there have been significant advances. It is becoming increasingly important for the physician, particularly surgeon, to be familiar with the indications and shortcomings of these compounds. Currently available tissue adhesives can be categorized as either fibrin tissue adhesives or acrylate-based tissue adhesives, e.g. cyanoacrylates. Although fibrin tissue adhesives and acrylate-based tissue adhesives, e.g. cyanoacrylates, are often discussed under the general topic of tissue adhesives, these two substances have different indications and mechanisms of action. Fibrin tissue adhesives use naturally occurring substrates that are part of normal endogenous clotting mechanisms. In contrast, the adhesion achieved by acrylate-based tissue adhesives, e.g. cyanoacrylates, is a result of synthetic compounds not naturally occurring in the human or animal body. These two types of adhesives also have different clinical indications. Fibrin tissue adhesives are typically applied below the dermis as a biologic hemostat or as a sealant for use with skin grafts and flaps. Acrylate-based tissue adhesives, e.g. cyanoacrylates, have been used most successfully at the level of the epidermis for superficial skin closure (see Toriumi D M, Raslan W F, Friedman M, et al. “Histotoxicity of cyanoacrylate tissue adhesives.”, Arch Otolaryngol Head Neck Surg 1990; 116: 546-50).
The mechanism of action of fibrin tissue adhesives is best understood by reviewing basic blood coagulation physiology. During the normal clotting process, thrombin cleaves the large molecular weight protein fibrinogen into smaller fibrin subunits. These subunits then undergo both end-to-end and side-to-side polymerization. Factor XIII (plasma glutaminase), in the presence of calcium, enables the cross-linking of these polymerized subunits into a stable fibrin clot. Usually, fibrin tissue adhesives are packaged as two separate components that when mixed on the injured or surgical field simulate the interaction of these endogenous compounds and form the final fibrin clot. The first component is composed of fibrinogen, factor XIII, and calcium chloride, while the second component is made up of thrombin and an antifibrinolytic agent. Fibrin tissue adhesives have found several practical uses in surgery such as cardiac surgery, vascular surgery, plastic surgery, and reconstructive surgery as hemostatic agents as well as adhesives. They may also be used for the closure of both skin grafts and local skin flaps. There are a variety of applicators available to deliver fibrin tissue adhesives to the surgical field. The simplest is the sequential delivery of the first component and second component with two separate syringes. A dual-syringe applicator can also be used. Fibrin tissue adhesives have the advantage that they are biocompatible and biodegradable. They also show minimal tissue reactivity. However, fibrin tissue adhesives have the disadvantage that they are very expensive. In addition, fibrin tissue adhesives are not easy to handle. For example, the two components of the fibrin tissue adhesive have to be transported and stored deep frozen (e.g. at −20° C.). Thus, it is very important that the (distribution) cold chain is not interrupted. In addition, fibrin tissue adhesives have to be packed as two separate components to avoid fibrin clot formation before application. However, even after mixing, the processing time is very short before the fibrin clot formation is completed.
Acrylate-based tissue adhesives such as cyanoacrylates were first used in surgery 1959 when Coover (see Coover H W, Joyner F B, et al. “Chemistry and performance of cyanoacrylate adhesives.” J Soc Plast Eng 1959; 15: 413-7) discovered their inherent adhesive properties. Cyanoacrylates include, but are not limited to, methyl-2-cyanoacrylate, buytl-2-cyanoacrylate, and octyl-2-cyanoacrylate. These compounds have their greatest utility in surgery such as facial plastic and reconstructive surgery as an alternative to traditional suture closure. They have also been used to close superficial wounds. In contrast to fibrin tissue adhesives, which rely on the interaction of endogenous compounds, the cyanoacrylate tissue adhesives are synthetic compounds that do not naturally occur in the human or animal body. One method of synthesizing an alkyl cyanoacrylate monomer is by reacting alkyl cyanoacetate with paraformaldehyde to form an intermediate compound. Heat applied to this intermediate compound causes depolymerization, resulting in an alkyl cyanoacrylate monomer liquid distillate (Toriumi D M, Raslan W F, Friedman M, et al. “Histotoxicity of cyanoacrylate tissue adhesives.” Arch Otolaryngol Head Neck Surg 1990; 116: 546-50). Acrylate-based tissue adhesives, e.g. cyanoacrylates, are less expensive than fibroin tissue adhesives. However, acrylate-based tissue adhesives, e.g. cyanoacrylates, have been shown to be histotoxic when applied below the dermis. For example, when applied below the skin, cyanoacrylates may induce histotoxicity as a result of biodegradation of the polymer into cyanoacetate and formaldehyde. In addition, acrylate-based tissue adhesives have the disadvantage that they seal the treated area tight so that cells can not enter this area to reconnect the separated tissues.
Thus, there is a need for tissue adhesives which overcome the afore-mentioned problems.
The inventors of the present invention surprisingly found that self-assembling polypeptides, particularly silk polypeptides such as spider silk polypeptides, are ideal tissue adhesives as they meet the following criteria: sufficient binding strength, uncomplicated and time-independent application, tissue biocompatibility, no tissue reactivity such as allergic or inflammatory reactions, and reasonable costs. It is also remarkable that the adhesive effect is achieved without enzymatic and/or chemical reactions as described for fibrin-based and acrylate-based tissue adhesives. Further, when used as tissue adhesives, the self-assembling polypeptides, particularly silk polypeptides such as spider silk polypeptides, do not form an insurmountable barrier so that the glued sites can be entered and passed by cells to generate new tissue. Furthermore, the self-assembling polypeptides, particularly silk polypeptides such as spider silk polypeptides, have the advantage that they are flexible which reduces or even abolishes the tensile force acting on the affected area. In addition, in contrast to acrylate- and fibroin-based tissue adhesives, self-assembling polypeptides prevent or at least delay drying out of the glued tissues.
Systems for the recombinant production of self-assembling polypeptides, particularly silk polypeptides, e.g. spider silk polypeptides are known in the art. Particularly, systems for the recombinant production of spider silk polypeptides in E. coli have been developed in WO 2006/008163 and WO 2006/002827. As an example, it is referred to WO 2006/008163 (claiming priority of U.S. provisional application No. 60/590,196). In this expression system, single building blocks (=modules) can be varied freely and can thus be adapted to the requirements of the specific case. Modules of this type are disclosed also in Hummerich et al., “Primary structure elements of dragline silks and their contribution to protein solubility and assembly”, 2004, Biochemistry 43, 13604-13612. Further modules are described in WO 2007/025719.